Decreasing potential iatrogenic risks associated with influenza vaccines

ABSTRACT

Influenza viruses for use in preparing human vaccines have traditionally been grown on embryonated hen eggs, although more modern techniques grow the virus in mammalian cell culture e.g. on Vero, MDCK or PER.C6 cell lines. The inventor has realised that the conditions used for influenza virus 5 culture can increase the risk that pathogens other than influenza virus may grow in the cell lines and have identified specific contamination risks. Suitable tests can thus be performed during manufacture in order to ensure safety and avoid iatrogenic infections.

All documents cited herein are incorporated by reference in theirentirety.

TECHNICAL FIELD

This invention concerns the production and quality control of influenzavirus vaccines.

BACKGROUND ART

Influenza viruses for use in preparing human vaccines have traditionallybeen grown on embryonated hen eggs, although more modern techniques growthe virus in mammalian cell culture e.g. on Vero cells, MDCK cells orPER.C6 cells. The change in virus growth substrate has provided anopportunity for regulatory re-assessment of influenza vaccine safety.For example, contamination with host cell DNA has been a regulatoryconcern for the cell-derived vaccines [1], but has not been of concernin the past for vaccines grown in eggs.

The safety issues surrounding egg-derived influenza vaccines are thusdifferent from those surrounding vaccines grown in cell culture, withcell-derived vaccines being under closer scrutiny. It is an object ofthe invention to address these different safety issues, and inparticular to provide methods for enhancing the safety of influenzavaccines grown on cell culture.

DISCLOSURE OF THE INVENTION

By definition, the use of mammalian cell substrates for influenzavaccine production involves culturing the cells under conditions thatare well suited to viral growth and replication. The inventor hasrealised that these conditions increase the risk that pathogens otherthan influenza virus may grow in the cell culture, thereby leading topotential contamination of the final vaccine product. Tests forcontamination are generally not difficult to perform, but a manufacturerfirst has to know what tests to perform. The inventor has identifiedspecific contamination risks, and their work means that suitable testscan be performed during manufacture in order to ensure the safety andquality of influenza vaccines grown on cell culture. Some of thecontaminants may be harmless in a final vaccine product, but theirpresence can interfere with influenza virus propagation and downstreampurification, and so their removal is primarily of concern for qualityand reproducibility, other contaminants would be harmful in a finalvaccine, and so their removal is primarily a safety concern.

The risk of contamination arising from viral co-culture is not withoutprecedent (e.g. certain early poliovirus vaccine batches werecontaminated by simian virus 40 (‘SV40’), a polyomavirus), but therehave not been any previous disclosures on identifying specific risksassociated with cell culture for human influenza vaccine production.Influenza viruses grown on cell culture are at particular risk fromcontamination because the strains used for vaccine production arechanged every year, and so new cultures have to be established everyyear. This annual change in production materials means that every newyear brings a new risk of contamination, particularly as multiplepassages are involved during preparation of seed viruses formanufacturers, thereby increasing the risk of parallel growth ofadventitious pathogenic agents.

The inventor has identified infectious agents that can grow in theconditions used for growing influenza viruses in cell culture but thatdo not grow in hen eggs. These infectious agents represent a newcontamination risk for influenza vaccines that was never of concern fortraditional influenza vaccines. Thus the invention provides a processfor preparing an influenza vaccine from influenza virus that has beengrown in a culture of a mammalian cell line, comprising a step in whichthe vaccine and/or the culture is tested for the presence of aninfectious agent that can grow in said cell line but that does not growin embryonated hen eggs.

The inventor has also identified infectious agents that grow in somecell substrates used for influenza vaccine production but do not grow inothers. These infectious agents are thus a contamination risk only forcertain influenza vaccines. Thus the invention also provides a processfor preparing an influenza vaccine from influenza virus that has beengrown in a culture of a first mammalian cell line, comprising a step inwhich the vaccine and/or the culture is tested for the presence of aninfectious agent that can grow in said first cell line but that does notgrow in a second mammalian cell line.

The invention also provides a process for preparing an influenza vaccinefrom influenza virus that has been grown in a culture of a mammaliancell line, comprising a step in which the vaccine and/or the culture istreated to remove and/or inactivate an infectious agent that can grow inthe cell line but does not grow in embryonated hen eggs. Similarly, theinvention provides a process for preparing an influenza vaccine frominfluenza virus that has been grown in a culture of a first mammaliancell line, comprising a step in which the vaccine and/or the culture istreated to remove and/or inactivate an infectious agent that can grow insaid first cell line but does not grow in a second mammalian cell line.After removal and/or inactivation, the vaccine/culture may be tested forthe presence of the infectious agent e.g. to verify that it is has beenremoved/inactivated.

The invention also provides an influenza vaccine that has been obtainedby a process of the invention. The invention also provides an influenzavaccine that is obtainable by a process of the invention.

The invention also provides an influenza vaccine that has been grown ina culture of a mammalian cell line, wherein the vaccine has beenconfirmed as free from the presence of an infectious agent that can growin said cell line but that does not grow in embryonated hen eggs.Similarly, the invention provides an influenza vaccine that has beengrown in a culture of a first mammalian cell line, wherein the vaccinehas been confirmed as free from the presence of an infectious agent thatcan grow in said first cell line but that does not grow in a secondmammalian cell line.

The invention also provides an influenza vaccine in which mammalianreovirus is undetectable by RT-PCR (e.g. using the L1-based RT-PCRtechnique disclosed in reference 16, using primers L1.rv5, L1.rv6,L1.rv7 and LV1.rv8 as taught). Not having been grown on eggs, thevaccine will be free from ovalbumin and from chicken DNA.

The Mammalian Cell Line

The influenza vaccines of the invention are grown in mammalian celllines, rather than being grown in embryonated eggs. Typical mammaliancell lines used in production of biologicals include: MDCK; CHO; BHK;Vero; MRC-5; PER.C6; WI-38; etc. Preferred mammalian cell lines forgrowing influenza viruses include: MDCK cells [2-5], derived from MadinDarby canine kidney; Vero cells [6-8], derived from African green monkey(Cercopithecus aethiops) kidney; or PER.C6 cells [9], derived from humanembryonic retinoblasts.

These cell lines are widely available e.g. from the American Type CellCulture (ATCC) collection [10], or from the Coriell Cell Repositories[11]. For example, the ATCC supplies various different Vero cells undercatalog numbers CCL-81, CCL-81.2, CRL-1586 and CRL-1587, and it suppliesMDCK cells under catalog number CCL-34.

As well as being useful for material derived from growth on mammaliancell lines, the invention can also be extended for material derived fromgrowth on avian cell lines [e.g. refs. 12 & 13], including cell linesderived from hens e.g. chicken embryo fibroblasts (CEF), etc.

Infectious Agents that do not Grow in Embryonated Hen Eggs

The inventor has identified a variety of pathogens that can grow inmammalian cell lines (in particular in both MDCK cells and Vero cells)used for preparing influenza virus for vaccine production but that donot grow in hen eggs. Testing for contamination by these pathogens wasnot necessary for vaccines prepared on the traditional egg substrate,but the inventor has realised that vaccine quality control shouldinclude tests for one or more of these pathogens in order to ensure thehighest safety standards. The pathogens are as follows:

-   -   Pneumovirinae, such as the Pneumovirus genus, including        respiratory syncytial virus (RSV).    -   Morbilliviruses of the Paramyxoviridae family, such as measles        virus.    -   Enteroviruses of the Picornaviridae family, such as Coxsackie        viruses, echoviruses and enteroviruses, although some Coxsackie        viruses (e.g. B3, B4) have been found not to grow in MDCK cells.    -   Mammalian Reoviridae, in particular orthoreoviruses (e.g.        mammalian reoviruses) and rotaviruses. Reoviruses can show        unrestricted growth in Vero and MDCK cells, and so testing for        them is of particular importance. Rotaviruses share the protease        requirements of influenza viruses in order to grow in cell        culture, and this parallel could unwittingly lead to activation        of contaminating rotaviruses.

Where these pathogens have different strains that have different hosts(e.g. human RSV and bovine RSV), the test will typically concernstrain(s) that can infect humans.

Testing for these agents is particularly important for viral strainsderived by reverse genetics techniques, as seed viruses for virusmanufacture will have undergone multiple passages in mammalian cellculture during the reverse genetics procedure, thereby increasing therisk of contamination by adventitious infective agents.

Infectious Agents that do not Grow in Eggs, but Grow in DifferentMammalian Cell Lines

The inventor has identified a variety of pathogens that do not grow inhen eggs, do not grow in MDCK cells, but do grow in Vero cells. Testingfor contamination by these pathogens was not necessary for vaccinesprepared on the traditional egg substrate and is not necessary forvaccines prepared on MDCK cells, but the inventor has realised thatquality control of vaccines grown on Vero cells should include tests forone or more of these pathogens in order to ensure the highest safetystandards. The pathogens are as follows:

-   -   Metapneumoviruses of the Paramyxoviridae family, such as human        metapneumovirus (HMPV).    -   Rubulaviruses of the Paramyxoviridae family, such as mumps        virus, which grows well in Vero.    -   Togaviridae, such as Rubellavirus.    -   Coronaviridae, such as the SARS coronavirus and other human        coronaviruses. These viruses show high growth levels in Vero        cells, with the SARS virus showing unrestricted growth, and so        testing for them is of particular importance.

Rhinoviruses of the Picornaviridae family, such as M-strains ofRhinovirus.

-   -   Varicella Zoster virus (VZV), also known as human herpes virus 2        (HHV3). VZV can show unrestricted growth in Vero cells, and so        testing for it is of particular importance.    -   Polyomaviridae, such as the SV-40 polyomavirus, the BK        polyomavirus and the JC polyomavirus. These polyomaviruses can        show unrestricted growth in Vero cells (particularly BK cells),        and so testing for them is of particular importance.    -   Porcine circoviruses.    -   Porcine picornaviruses, such as swine vesicular disease virus        (SVDV) and Teschen-Talfan virus.    -   Chlamydia bacteria, including C. trachomatis, C. pneumoniae        and C. psittaci. These bacteria may grow in Vero cells, and so        testing for them is of particular importance.    -   Parvoviruses such as canine parvovirus (CPV) or porcine        parvoviruses.

Where these pathogens have different strains that have different hosts(e.g. human RSV and bovine RSV), the test will typically concernstrain(s) that can infect humans.

Testing for non-human viruses (e.g. avian and porcine viruses) is mainlyof concern only when avian or porcine materials have been used in viralpreparation e.g. if strains were initially isolated from pigs or birds,or if egg passages were used during initial growth, or if porcinetrypsin was used in cell culture, etc.

Infectious Agents that Grow in Eggs and Mammalian Cell Lines

The inventor has also identified pathogens that, in contrast to thosedescribed above, grow both in mammalian cell lines and in hens eggs. Aprocess of the invention may involve a step of testing for suchpathogens, but this step would also be part of enhanced quality controlof viruses grown in hens eggs. These pathogens include:

-   -   Parainfluenza viruses (PIV), members of the Paramyxoviridae        paramyxovirinae, including PIV-1, PIV-2 and PV-3.    -   The Herpesviridae, such as herpes simplex virus 1 and 2.    -   The Adenoviridae, such as the adenoviruses, including human and        simian adenovirus.    -   Mycoplasma.    -   Avian circoviruses.    -   Avian Reoviridae, in particular orthoreoviruses, such as avian        reoviruses that can grow in mammalian cell lines.

The inventor has also identified pathogens that grow in hen eggs and inVero cells, but do not more unlikely to grow in MDCK cells. A process ofthe invention may involve a step of testing for such pathogens, but thisstep would also be part of enhanced quality control of viruses grown inhens eggs, and the step is not necessary if a MDCK substrate is used.These pathogens include:

-   -   Birnaviridae, such as infectious bursal disease virus (also        known as gumboro virus).

Testing for agents that grow in both eggs and cell lines is importantfor viral strains derived after multiple passages in eggs e.g. seedviruses for virus manufacture.

As these pathogens grow in eggs then testing for their presence can alsobe used for viruses prepared from viruses grown on eggs. Thus theinvention is not limited to vaccines grown on cell culture, but can alsobe used for ‘traditional’ egg-based vaccines.

Testing Methods

Methods for detecting the presence of pathogens in cell cultures and inbiopharmaceuticals are routinely available. Methods will generally relyon immunochemical detection (immunoassay, western blot, ELISA, etc.)and/or on nucleic acid detection (hybridisation methods, such a Southernblots or slot blots, PCR, etc.). As an alternative, it is possible totest for the presence of a pathogen by conventional cell cultureinoculation (i.e. test whether the material leads to production of thecontaminating pathogen when cultured under suitable conditions).

Methods may detect a single pathogen (e.g. virus) or may detect multiplepathogens (e.g. viruses) Where a test detects multiple pathogens (e.g.‘X’, ‘Y’ or ‘Z’) then it may give a specific result (e.g. virus ‘Y’ ispresent) or it may give a general result (e.g. one of ‘X’, ‘Y’ or ‘Z’ ispresent). Methods may be quantitative, semi-quantitative or qualitative.Real-time detection methods may be used.

General guidance for detecting a pathogen (e.g. virus) of interest canbe found in reference 14. A number of more specific assays are given inthe following paragraph, and the skilled person can readily find orprepare an assay for detecting the presence of any chosen pathogen.

Reference 15 discloses a multiplex reverse transcription PCR (RT-PCR)assay, referred to as ‘m-RT-PCR-ELISA’, for the detection of ninerespiratory tract pathogens in a single test, namely: enterovirus,influenza virus type A and type B, respiratory syncytial virus,parainfluenzavirus type 1 and type 3, adenovirus, Mycoplasma pneumoniaeand Chlamydia pneumoniae. A RT-PCR method for detecting mammalianreovirus is disclosed in reference 16. Reference 17 discloses areal-time RT-PCR assay for detecting human metapneumoviruses from allknown genetic lineages. Reference 18 discloses a single RT-PCR assay fordetection of human respiratory syncytial virus (HRSV), humanparainfluenzaviruses 1, 2, & 3 and influenza A & B. Reference 19discloses a multiplex RT-PCR assay to detect and differentiate measlesvirus, rubella virus, and parvovirus B19. A real-time RT-PCR assay todetect human rhinovirus with accurate discrimination from other virusesfrom the family Picornaviridae is disclosed in reference 20. Reference21 discloses a multiplex RT-PCR assay with nested primer sets targetedto conserved regions of human parainfluenza virus haemagglutinin, humancoronavirus spike protein, and human enterovirus and rhinoviruspolyprotein genes, which permits rapid, sensitive, and simultaneousdetection and typing of the four types of parainfluenza viruses (1, 2,3, 4AB), human coronavirus 229E and OC43, and the generic detection ofenteroviruses and rhinoviruses. SVDV detection by RT-PCR is disclosed inreference 22. A one step quantitative RT-PCR assay for the SARScoronavirus is disclosed in reference 23. Reference 24 discloses aTaqMan allelic discrimination real-time PCR assay for VZV. A multiplexPCR assay for rapid simultaneous detection of pseudorabies viruses,parvoviruses and circoviruses is disclosed in reference 25. A real-timeFRET probe PCR assay for SV-40 polyomavirus detection is described inreference 26. Reference 27 discloses an assay for simultaneous detectionand differentiation of human polyomaviruses JC and BK by a rapid andsensitive PCR-ELAHA method. Detection of porcine circoviruses in humancell lines by PCR and indirect immune fluorescence assays is disclosedin reference 28. PCR methods for birnavirus detection are disclosed inreferences 29 & 30.

The detection method of the invention may be performed at any stage(s)during vaccine manufacture starting from the seed virus and/or the cellsubstrate and/or the culture medium, through the viral infection andgrowth stages, through viral harvest, through any viral processing (e.g.splitting and/or surface protein extraction), through vaccineformulation and then to vaccine packaging. Thus the assay used accordingto the invention can be performed on the materials used to create thevital culture, on the viral culture itself, and on material extractedand derived from the viral culture. The assay need not be performed oneach and every vaccine or culture, but can be used at appropriateintervals as part of normal quality control. It is particularly usefulwhen vaccine production is changed for the new yearly strainsrecommended by regulatory authorities, at which stage new cultures areestablished and must be subjected to new quality control. Assays of theinvention are advantageously performed on the seed virus used forvaccine manufacture.

In the methods of the invention, the cell lines used to grow influenzaviruses may be cultured in any suitable medium e.g. in serum-free media,in protein-free media, etc. Methods for the serum-free culture ofinfluenza virus are disclosed in reference 2, and methods forprotein-free culture are disclosed in reference and/or protein-free 31.A “protein-free” medium may, however, include one or more proteases(e.g. trypsin) that may be necessary for influenza virus propagation. Aserum-free medium may include serum supplements.

It is also preferred that the vaccine should have been grown in aculture without the addition of bovine-derived material, therebyensuring that the culture is free from any possible BSE contaminationand from bovine viruses. Media that do not include components associatedwith any transmissible spongiform encephalopathy are preferred.

The Influenza Vaccine

The invention concerns quality control of influenza vaccines. Thevaccine may be in the form of a live virus or, preferably, aninactivated virus. Virus inactivation typically involves treatment witha chemical such as formalin or β-propiolactone. Where an inactivatedvirus is used, the vaccine may be a whole virus, a split virus, or viralsubunits. Split viruses are obtained by treating virions with detergents(e.g. ethyl ether, polysorbate 80, deoxycholate, tri-N-butyl phosphate,Triton X-100, Triton N101, cetyltrimethylammonium bromide, etc.) toproduce subvirion preparations. Subunit vaccines comprise the influenzasurface antigens haemagglutinin and neuraminidase. Influenza antigenscan also be presented in the form of virosomes [32].

Influenza vaccines of the invention can be based on any suitablestrain(s). Vaccines typically include antigens from at least one strainof influenza A virus and/or at least one strain of influenza B virus.The recommended strains for vaccines change from season to season. Inthe current inter-pandemic period, vaccines typically include twoinfluenza A strains (H1N1 and H3N2) and one influenza B strain, andtrivalent vaccines are preferred. The invention is also suitable forpreparing viruses from pandemic strains, such as H5 or H7 strains, thatis strains to which the human population is immunologically naïve.Vaccines in pandemic situations may be monovalent, or they may be basedon a normal trivalent vaccine supplemented by a pandemic strain.

The influenza virus(es) used in the processes of the invention may bereassortant strains, and/or may have been obtained by reverse geneticstechniques. The virus(es) may be attenuated. The virus(es) may betemperature-sensitive. The virus(es) may be cold-adapted.

Where a vaccine includes more than one strain of influenza, thedifferent strains are typically grown separately and are mixed after theviruses have been harvested and antigens have been prepared. Thus theprocesses of the invention may include the step of mixing antigens frommore than one influenza strain. Testing for pathogens may be performedbefore or after such mixing.

The vaccine will typically be prepared for administration to a patientby injection (e.g. subcutaneous injection or intramuscular injection),although other routes of administration are known for influenza vaccinese.g. intranasal [33-35], oral [36], intradermal [37,38], transcutaneous,transdermal [39], etc.

Vaccines prepared according to the invention may be used to treat bothchildren and adults. Influenza vaccines are currently recommended foruse in pediatric and adult immunisation, from the age of 6 months.Safety concerns are most acute for pediatric vaccines, particularly asimmunologically naive subjects typically receive two vaccine doses in ashort period (e.g. at a 1 or 2 month interval).

Vaccines of the invention may include an adjuvant. Adjuvants that havebeen used in influenza vaccines include aluminium salts [40,41],chitosan [42], CpG oligodeoxynucleotides such as CpG 7909 [43],oil-in-water emulsions such as MF59 [44], water-in-oil-in-wateremulsions [45], E. coli heat labile toxin [34,46] and its detoxifiedmutants [47-48], monophosphoryl lipid A [49] and its 3-o-deacylatedderivative [50], pertussis toxin mutants [51], muramyl dipeptides [52],etc.

Haemagglutinin (HA) is the main immunogen in inactivated influenzavaccines, and vaccines doses are standardised by reference to HA levels,typically as measured by a single radial immunodiffution (SRID) assay.Vaccines typically contain about 15 μg of HA per strain, although lowerdoses are also used e.g. for children, or in pandemic situations.Fractional doses such as ½ (i.e. 7.5 μg HA per strain), ¼ and ⅛ havebeen used [40,53]. Thus vaccines may include between 1 and 20 μg of HAper influenza strain, preferably e.g. about 15, about 10, about 7.5,about 5, about 3.8, about 1.9, etc.

The vaccines may include preservatives such as thiomersal or2-phenoxyethanol. It is preferred, however, that the vaccine should besubstantially free from (i.e. less than 5 μg/ml) mercurial material e.g.thiomersal-free [54,55]. Vaccines containing no mercury are morepreferred.

The vaccines of the invention preferably contain less than 10 ng(preferably less than 1 ng, and more preferably less than 100 pg) ofresidual host cell DNA per dose, although trace amounts of host cell DNAmay be present. Contaminating DNA can be removed during vaccinepreparation using standard purification procedures e.g. chromatography,etc. Removal of residual host cell DNA can be enhanced by nucleasetreatment e.g. by using the Benzonase™ DNase [1]. Vaccines containing<10 ng (e.g. <1 ng, <100 pg) host cell DNA per 15 μg of haemagglutininare preferred, as are vaccines containing <10 ng (e.g. <1 ng, <100 pg)host cell DNA per 0.25 ml volume. Vaccines containing <10 mg (e.g. <1ng, <100 pg) host cell DNA per 50 μg of haemagglutinin are morepreferred, as are vaccines containing <10 ng (e.g. <1 ng, <100 pg) hostcell DNA per 0.5 ml volume.

These various characteristics of the vaccines may be achieved byincluding suitable steps in the processes of the invention. Specificsteps thus include: a step of inactivation; a step of mixing three virusstrains to make a trivalent vaccine; a step of formulating the vaccinefor injection; a step of administering the vaccine to a patient; a stepof combining the vaccine with an adjuvant; a step of measuring HAcontent; a step of adjusting HA content e.g. by dilution; a step ofadding a preservative; a step of removing residual host cell nucleicacids; etc.

Preferred Adventitious Agents for Testing

Preferred embodiments of the invention involve adventitious agents, andparticularly viruses, that are found in respiratory samples, as theseare more likely to be present in initial clinical isolates of influenzavirus. Respiratory pathogens include: RSV, PIV-3, SARS coronavirus,adenoviruses, rhinoviruses, reovirus (‘respiratory enteritic orphanvirus’), etc. Herpes simplex virus can also be found in respiratorysamples.

Particularly preferred pathogens for which the invention is used are:reoviruses (particularly mammalian reoviruses); polyomaviruses;birnaviruses; circoviruses; and parvoviruses. Testing for herpes simplexviruses is also preferred.

Where a vaccine has been treated with detergent (e.g. a split or asubunit vaccine) then this treatment step offers an extra degree ofsafety, as it can also disrupt the contaminating viruses. If thecontaminant is non-enveloped, however, then the detergent treatment willusually have no effect on the vaccine, and so it does not itself improvesafety. Thus testing for the following pathogens is particularlyimportant, as they are non-enveloped: Picornaviridae, Reoviridae,Birnaviridae, Parvoviridae, Circoviridiae, Adenoviridae, Polyomaviridae.

Detergent resistance of these viruses combined with their high growth inVero cells means that it is particularly important to test for the humanenteroviruses, the mammalian Reoviridae, the Adenoviridae and thePolyomaviridae when using a Vero cell substrate. The mammalianReoviridae also grow at high levels in MDCK cells. These viruses arealso among those most resistance to inactivation.

Testing for the presence of mammalian Reoviridae is a preferredembodiment of the invention, as: (a) the viruses do not readily grow inhen eggs, and so testing for them has not been part of traditionalinfluenza virus manufacture; (b) the viruses can show unrestrictedgrowth in both MDCK and Vero cell lines; (c) the viruses are highlyresistant to inactivation and remain stable during vaccine processing;(d) the viruses are non-enveloped and so can survive detergent treatmentof influenza virus; and (e) the viruses are involved in respiratoryinfections and so could contaminate initial viral isolates. Testing foravian Reoviridae is also important where avian materials have been usedduring preparation of the virus, and criteria (b) to (e) listed aboveapply equally to avian reoviruses.

Other Biologicals

As well as being useful for testing influenza vaccines, the inventioncan also be used for other biologicals, such as recombinant proteinse.g. antibodies [56], growth factors, cytokines, lymphokines, receptors,hormones, vaccine antigens, diagnostic antigens, etc.

General

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The term “about” in relation to a numerical value x means, for example,x±10%.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

Further general information on influenza vaccines, including strains,cell lines for growth, doses, combinations, formulations, etc. can befound in chapters 17 & 18 of reference 57. Further details on influenzavirus, including details of its life cycle during viral growth, can befound in chapter 46 of reference 14.

MODES FOR CARRYING OUT THE INVENTION MDCK Cells

The inventor has extensive experience of growing influenza viruses onMDCK cells in serum-free culture for the preparation of vaccines. Theyhave realised that the cells are also suitable hosts for otherpathogenic agents, and so the ability of various other pathogens to growin the same conditions was tested (specifically, culture of MDCK 33016,deposited as DSM ACC2219, in serum-free medium, as disclosed inreference 2).

When testing for active virus replication or growth in MDCK cells, testsfor respiratory syncytial viruses RSV-A2 and RSV-B were negative.Parainfluenzavirus strains PI-3 and SV-5 were detected. Tests for humancoronaviruses 229E and SARS were negative, as were tests for poliovirusI, echovirus 6, coxsackievirus A16 and coxsackievirus B3. Type Ib, 37and NL.9501841 rhinoviruses tested negative. Tests for reovirus Reo3were positive, as were tests for herpes simplex virus HSV-1. Tests forhuman adenoviruses 1, 5 and 6 were negative. SV-40 tests were negative,and inoculum titers were stable for 14 days. Canine parvovirus andminute virus of mice tested negative, as did Rous sarcoma virus.Mycoplasma hyorhinis tested negative. Chlamydia trachomatis testednegative, although a very low level of growth could not be excludedduring days 3-5 after infection.

Further investigation revealed that MDCK cells can support growth ofvesicular stomatitis (Indiana) virus, vaccinia virus, coxsackievirus B5;reovirus 2; human adenovirus types 4 and 5; vesicular exanthema of swinevirus, and infectious canine hepatitis virus [58].

Of the viruses which could be grown in MDCK cells, parainfluenzaviruses,herpes simplex viruses and adenoviruses can also grow in embryonated heneggs. In contrast, the human reoviruses (and other mammalian reoviruses)do not readily grow in eggs. If MDCK is used as a cell culture systemfor influenza virus production, therefore, quality control testingshould check for contamination by human reoviruses. The inventorestimates that reovirus levels could increase by 5 logs or noose duringrepeated passages in MDCK suspension cultures, whereas levels of a virussuch as adenovirus would decrease by 6 to 10 logs. Herpes simplex viruslevels should also be checked, as HSV growth of at least 8 logs ispossible. Similarly, PIV-3 growth of 8 logs has been seen after 1 weekof culture.

Vero Cells

Following the testing work on MDCK cells, replication of pathogens inVero cells was investigated. Vero cells support the growth of pathogenssuch as: pneumoviruses, such as RSV-A and RSV-B; human metapneumoviruses(HMPV); morbilliviruses, such as measles virus; paramyxoviruses such asmumps virus and parainfluenza virus; rubellavirus; human and aviancoronaviruses; picornaviruses, such as entroviruses, echoviruses andcoxsackie viruses, and porcine SVDV and Teschen-Talfan virus; mammalianand avian reoviruses; herpesviruses, such as HSV-1 and HSV-2; simian andhuman adenoviruses; varicella zoster virus (VZV); polyomaviruses, suchas JC, BK and SV-40; birnaviruses, such as gumborovirus; porcinecircoviruses; canine parvovirus; and Chlamydia.

Of these pathogens, the following do not grow in hen eggs, and are thusnew risks for contamination of influenza vaccines when Vero cells areused as a substrate: RSV; HMPV; measles virus; rubellavirus; humancoronaviruses; enteroviruses; reoviruses; VZV; polyomaviruses; porcinepicornaviruses, parvoviruses and circoviruses. Many of these pathogensdo not grow in MDCK cells, showing that MDCK is a safer substrate forinfluenza vaccine production. Emerging viruses such as the SARScoronavirus grow on Vero cells, but not on MDCK cells. Similarly, VZVgrows on Vero cells, but not on hen eggs or on MDCK cells. Vaccinationwith a Vero-derived influenza vaccine that was inadvertentlycontaminated with this coronavirus or with VZV could lead to aniatrogenic outbreak of SARS and/or chickenpox, which would be disastrousboth to the population and to the reputation of vaccines. Havingidentified these risks, however, appropriate quality control mechanismscan be put in place.

In addition to Vero cells, PER.C6 cells support growth of adenoviruses[59,60]. Based on known viral characteristics, PER.C6 cells can also beexpected to support the growth of at least parainfluenzaviruses andreoviruses.

It will be understood that the invention is described above by way ofexample only and modifications may be made while remaining within thescope and spirit of the invention.

REFERENCES The Contents of Which are Hereby Incorporated by Reference

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1. A process for preparing an influenza vaccine from influenza virusthat has been grown in a culture of a mammalian cell line, comprising astep in which the vaccine and/or the culture is tested for the presenceof an infectious agent that can grow in said cell line but that does notgrow in embryonated hen eggs.
 2. A process for preparing an influenzavaccine from influenza virus that has been grown in a culture of amammalian cell line, comprising a step in which the vaccine and/or theculture is treated to remove and/or inactivate an infectious agent thatcan grow in the cell line but does not grow in embryonated hen eggs. 3.The process of claim 1 or claim 2, wherein the mammalian cell line is aMDCK cell line, a Vero cell line, or a PER.C6 cell line.
 4. The processof any preceding claim, wherein the infectious agent is selected fromthe group consisting of: Pneumovirinae; Morbilliiviruses of theParamyxoviridae family; Enteroviruses of the Picornaviridae family;mammalian Reoviridae; and Birnaviridae.
 5. The process of claim 4,wherein the infectious agent is selected from the group consisting of:respiratory syncytial virus; measles virus; Coxsackie viruses;echoviruses; enteroviruses; orthoreoviruses; rotaviruses; and infectiousbursal disease virus.
 6. The process of any preceding claim, wherein themammalian cell line is a Vero cell line, and wherein the infectiousagent is selected from the group consisting of: Metapneumoviruses of theParamyxoviridae family; Rubulaviruses of the Paramyxoviridae family;Togaviridae; Coronaviridae; Rhinoviruses of the Picornaviridae family;varicella zoster virus; Polyomaviridae; porcine circoviruses; porcinepicornaviruses; Chlamydia bacteria; and Parvoviruses.
 7. The process ofclaim 6, wherein the infectious agent is selected from the groupconsisting of: human metapneumovirus; mumps virus; Rubellavirus; SARScoronavirus; M-strains of Rhinovirus; SV-40 polyomavirus; BKpolyomavirus; JC polyomavirus; swine vesicular disease virus;Teschen-Talfan virus; C. trachomatis; C. pneumoniae; C. psittaci; canineparvovirus; and porcine parvoviruses.
 8. The process of any precedingclaim, further comprising a step in which the vaccine and/or the cultureis tested for the presence of a pathogen selected from the groupconsisting of: parainfluenza viruses; Herpesviridae; Adenoviridae;Mycoplasma; avian circoviruses; and avian Reoviridae.
 9. A process forpreparing an influenza vaccine from influenza virus that has been grownin a culture of a first mammalian cell line, comprising a step in whichthe vaccine and/or the culture is tested for the presence of aninfectious agent that can grow in said first cell line but that does notgrow in a second mammalian cell line.
 10. A process for preparing aninfluenza vaccine from influenza virus that has been grown in a cultureof a first mammalian cell line, comprising a step in which the vaccineand/or the culture is treated to remove and/or inactivate an infectiousagent that can grow in said first cell line but does not grow in asecond mammalian cell line.
 11. The process of claim 9 or claim 10,wherein (a) the first mammalian cell line is selected from the groupconsisting of: a MDCK cell line, a Vero cell line, or a PER.C6 cellline; (b) the second mammalian cell line is selected from the groupconsisting of: a MDCK cell line, a Vero cell line, or a PER.C6 cellline; and (c) the first and second mammalian cell lines are different.12. The process of any one of claims 9 to 11, wherein the first cellline is a Vero cell line, and wherein the infectious agent is selectedfrom the group consisting of: Metapneumoviruses of the Paramyxoviridaefamily; Rubulaviruses of the Paramyxoviridae family; Togaviridae;Coronaviridae; Rhinoviruses of the Picornaviridae family; varicellazoster virus; Polyomaviridae; porcine circoviruses; porcinepicornaviruses; Chlamydia bacteria; Parvoviruses; and Birnaviridae. 13.The process of claim 12, wherein the infectious agent is selected fromthe group consisting of: human metapneumovirus; mumps virus;Rubellavirus; SARS coronavirus; M-strains of Rhinovirus; SV-40polyomavirus; BK polyomavirus; JC polyomavirus; swine vesicular diseasevirus; Teschen-Talfan virus; C. trachomatis; C. pneumoniae; C. psittaci;canine parvovirus; porcine parvoviruses; and infectious bursal diseasevirus.
 14. The process of any one of claims 9 to 13, further comprisinga step in which the vaccine and/or the culture is tested for thepresence of a pathogen selected from the group consisting of:parainfluenza viruses; Herpesviridae; Adenoviridae; Mycoplasma; aviancircoviruses; and avian Reoviridae.
 15. The process of claim 2 or claim10, comprising a further step wherein, after said removal and/orinactivation, the vaccine and/or culture is tested for the presence ofsaid infectious agent.
 16. A process for preparing an influenza vaccinefrom influenza virus that has been grown in a culture of a mammaliancell line or in hens eggs, comprising a step in which the vaccine and/orthe culture is tested for the presence of a pathogen selected from thegroup consisting of: parainfluenza viruses; Herpesviridae; Adenoviridae;Mycoplasma; avian circoviruses; avian Reoviridae; and Birnaviridae. 17.The process of claim 16, wherein the pathogen is selected from the groupconsisting of: PIV-1; PIV-2; PIV-3; herpes simplex virus 1; herpessimplex virus 2; human adenovirus; simian adenovirus; orthoreoviruses;and infectious bursal disease virus.
 18. The process of any precedingclaim, wherein the culture is tested by immunochemical detection and/ornucleic acid detection.
 19. The process of claim 18, wherein detectionis by ELISA and/or PCR (including RT-PCR).
 20. An influenza vaccine thathas been grown in a culture of a mammalian cell line, wherein thevaccine has been confirmed as free from the presence of an infectiousagent that can grow in said cell line but that does not grow inembryonated hen eggs.
 21. An influenza vaccine that has been grown in aculture of a first mammalian cell line, wherein the vaccine has beenconfirmed as free from the presence of an infectious agent that can growin said first cell line but that does not grow in a second mammaliancell line.
 22. The vaccine of claim 18 or claim 19, wherein vaccine wasgrown in a culture of a MDCK cell line, of a Vero cell line, or of aPER.C6 cell line.
 23. An influenza vaccine in which mammalian reovirusis undetectable by RT-PCR.
 24. The vaccine of claim 23, which is freefrom ovalbumin and from chicken DNA.
 25. An influenza vaccine obtainedor obtainable by the process of any one of claims 1 to
 17. 26. Thevaccine of any one of claims 20 to 25, which is a live virus vaccine.27. The vaccine of any one of claims 20 to 25, which is an inactivatedvirus vaccine.
 28. The vaccine of claim 27, which is a whole virusvaccine, a split virus vaccine, or a viral subunit vaccine.
 29. Thevaccine of any one of claims 20 to 28, which is a trivalent influenzavaccine.
 30. The vaccine of any one of claims 20 to 29, which includes apandemic influenza virus strain.
 31. The vaccine of claim 30, whichincludes a H5 or H7 influenza virus strain.
 32. The vaccine of any oneof claims 20 to 31, which is for administration to a patient byinjection, by an intranasal route, by an oral route, by an intradermalroute, by a transcutaneous route, or by a transdermal route.
 33. Thevaccine of any one of claims 20 to 32, which is for pediatricimmunisation.
 34. The vaccine of any one of claims 20 to 33, whichincludes 1-20 μg of influenza virus haemagglutinin per strain.
 35. Thevaccine of any one of claims 20 to 34, which includes an adjuvant.